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Tetrameric alpha-synuclein and use thereof

a technology of tetrameric alpha-synuclein and tetrameric alpha-synuclein, which is applied in the field of tetrameric alpha-synuclein, can solve problems such as toxic aggregation

Inactive Publication Date: 2013-10-31
BRANDEIS UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods and systems for identifying and characterizing compounds that decrease the levels and activity of tetrameric α-synuclein, which is a protein associated with certain neurodegenerative disorders. The invention also provides methods for stabilizing tetrameric α-synuclein and reducing its levels in the brain. The invention recognizes that increased expression levels of α-synuclein may increase the levels of free α-synuclein monomer, which can aggregate and lead to toxic effects. The invention proposes identifying and characterizing compounds that decrease the levels and activity of tetrameric α-synuclein and assessing their effects on the levels, stability, and activity of the protein. The invention also provides methods for inducing a conformational change in tetrameric α-synuclein to stabilize it.

Problems solved by technology

This finding is contrary to the previous reports and beliefs that α-synuclein exists as a disordered peptide prone to mutimerization under certain conditions, which results in toxic aggregation that forms the basis for certain neurodegenerative disorders.

Method used

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  • Tetrameric alpha-synuclein and use thereof
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  • Tetrameric alpha-synuclein and use thereof

Examples

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example 1

Isolation of an α-Synuclein Oligomer

[0209]We expressed α-synuclein as an N-terminal GST-fusion and have developed a purification procedure aimed at avoiding denaturing conditions and maintaining the protein in a ‘physiological-like’ condition, in buffer containing 100 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, and 0.1% n-octyl-glucopyranoside (BOG). We note that the concentration of BOG present (˜3 mM) is well below the critical micelle concentration (˜25 mM). The same buffer was used in all subsequent protein purification steps as well as for storage. After proteolytic removal of the GST tag, α-synuclein was purified to homogeneity on a size-exclusion column, from which it eluted as a single, sharp, and symmetrical peak suggesting a homogenous particle. The specific protease site required for GST tag removal leaves a ten-residue sequence, GPLGSPEFPG (SEQ ID NO: 5), prior to the N-terminal methionine of the canonical α-synuclein sequence. However, evidence from NMR resonance assign...

example 2

Characterization of Native α-Synuclein Tetramer

[0282]To identify the assembly form of αSyn under non-denaturing conditions and avoid the potential breakdown of physiological complexes by detergents, we employed native gel electrophoresis (Native-PAGE). Since αSyn is endogenously expressed in a variety of cultured cell types, we chose to analyze the dopaminergic human neuroblastoma line, M17D (10), as well as the commonly used cell lines HEK293, HeLa, and COS-7 that endogenously express αSyn. Each of these cell types contained a nondenatured αSyn-immunoreactive species migrating at ˜45 kDa in Blue Native PAGE (BN-PAGE). This was clearly the predominant form in all the cells, and very little or no ˜14 kDa monomer was detectable (FIG. 16A). We note that similar results were observed for heterologously expressed recombinant α-Syn that was purified from bacteria under non-denaturing conditions. Because these initial results in untransfected cells suggested an apparently stable oligomeric...

example 3

A Soluble α-Synuclein Construct Forms a Dynamic Tetramer

[0336]A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer...

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Abstract

Disclosed herein are methods and related compositions for identifying compounds that stabilize natively folded tetrameric α-synuclein. These methods and compositions are useful for the treatment and diagnosis of α-synuclein-associated diseases and disorders.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 61 / 410,860, filed Nov. 5, 2010, and U.S. Provisional Application Ser. No. 61 / 410,861, filed Nov. 5, 2010, the entire contents of each of which are hereby incorporated by reference.BACKGROUND[0002]The protein α-synuclein is associated with multiple neurological disorders, including the two most prevalent neurodegenerative diseases, Parkinson's disease and Alzheimer's disease. Collectively, these α-synuclein associated disorders are referred to as synucleinophathies, and most are characterized by the presence of insoluble α-synuclein-rich aggregates called Lewy bodies (1-3). The presence of Lewy bodies in neurons of the substantia nigra is the histopathological hallmark of Parkinson disease, and is currently used to differentiate Parkinson disease from other neurological disorders with overlapping clinical symptoms (4). In addition to α-synuclein be...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/68A61K45/00C07K16/18
CPCG01N33/6896C07K16/18A61K45/00G01N2800/2821G01N2800/2835G01N2800/52G01N33/6893
Inventor RINGE, DAGMARPETSKO, GREGORY A.SELKOE, DENNIS J.BARTELS, TIM
Owner BRANDEIS UNIV
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